PROJECT SUMMARY Genetic analysis in model organisms relies on tools to inactivate genes in particular cells at specific times. Most existing methods for gene inactivation, such as conditional gene deletion or RNAi, target DNA or mRNA. However, phenotypes do not become evident until pre-existing protein product from the targeted gene has decayed. This lag can be many hours or even days. However, in many cases, it is essential to rapidly inactivate genes, such as when performing experiments in developing organisms, or when studying a gene that produces a cell lethal phenotype when removed. In an attempt to circumvent these limitations, several strategies have been created that target protein gene product directly, typically by tagging the protein with a degron that can be induced to degrade the tagged protein. However, these methods are poorly suited for the study of rapidly occurring developmental events, as they either work slowly (several hours), necessitate the addition of drugs that may be difficult to introduce into embryos, or require the prolonged illumination of specific cells with light. We recently developed a degron-based method in C. elegans, called ZF1-tagging, that very rapidly removes tagged proteins to reveal loss-of-function phenotypes. Proteins tagged with the ZF1 degron can be induced to degrade by expressing the adaptor ZIF-1, which binds the ZF1 domain and targets the tagged protein to a conserved E3 ubiquitin ligase complex. In its current form, ZF1-tagging can be used to degrade proteins with either spatial or temporal control, but not both. Here we propose to engineer significant improvements to the ZF1-tagging system. Specifically, we will (1) expand ZF1-tagging so that it can be used to degrade proteins with combined spatial and temporal control; (2) adapt ZF1-tagging to rapidly kill cells genetically; and (3) engineer ZF1-tagging to function in zebrafish, where an effective genetic tool for inactivating genes in specific tissues is lacking. These improvements will make ZF1-tagging an extremely powerful and versatile system for inactivating genes rapidly in specific cells at specific times in model organisms, and would provide proof-of-principle that the method could be adapted to function in any system.